Estrogen Signaling Modulation Prevents and Even Reverses Skeletal Muscle Fibrosis

Abstract

Background: Skeletal muscle fibrosis represents accumulation of extracellular matrix (ECM), often leading to muscle weakness and atrophy. Notably, lower abdominal muscle (LAM) fibrosis and atrophy cause inguinal herniation—a prevalent condition lacking pharmacological treatment. We developed a herniation mouse model, Aromhum, characterized by spontaneous scrotal hernias due to local estradiol (E2) production within LAM as well as hernia-associated fibroblasts (HAFs) that express platelet-derived growth factor alpha (PDGFRA) and estrogen receptor alpha (ESR1). Objectives & Hypothesis: Our objective was to investigate the mechanism of ESR1 signaling on LAM HAFs and subsequent development of hernias. Methods: We manipulated estrogen signaling in two ways: first, we generated fibroblast-specific estrogen receptor alpha knockout mice (fEsr1−/−-Aromhum) capable of local E2 production but incapable of signaling through ESR1 (n = 5-10 mice/group). Second, we blocked E2 signaling pharmacologically using the potent ESR1 antagonist fulvestrant (0.15mg/kg, 90 days, n=10-15 mice/group). We conducted in vitro experiments on primary LAM HAFs exposed to 10nM E2 ± 100nM Fulvestrant (24-48h). These cells underwent analysis via ESR1 ChIP, ATAC, and RNA-seq in 3 technical replicates (3-5 mice each). Additionally, to demonstrate clinical relevance, we probed human herniated LAM tissues from patients (n=25 samples, 21-76 years). Results: All Aromhum mice develop scrotal hernias by 6 weeks. However, fEsr1−/−-Aromhum mice did not develop hernias, while littermate controls exhibited hernias. Thus, HAF ESR1 depletion mitigated LAM fibrosis and atrophy. Similarly, administering fulvestrant prior to hernia onset prevented hernia development. Remarkably, fulvestrant treatment in mice that had developed large scrotal hernias (>200mm2) led to complete hernia regression with reversed muscle fibrosis and fiber atrophy (collagen levels comparable to wild-type mice). In vitro HAF culturing and subsequent multiomic analyses unveiled a core set of 58 genes directly influenced by E2/ESR1, including crucial ECM genes like fibulins ( Fbln5, Fbln7), metalloproteases ( Adamts3, Adamts6), and signaling molecules ( Ltbp1, Ncam1, Piezo2). Pathways such as TGFβ, WNT, and N-Glycan biosynthesis were significantly upregulated by E2. Human herniated LAM tissues exhibited substantial fibrosis, along with stromal HAF markers PDGFRA and ESR1. We also validated expression of the core E2-modulated genes in human LAM tissues ( NCAM1, LTBP1, ADAMTS6, PIEZO2). Collectively, these findings provide compelling evidence of ESR1 pathway activation and downstream gene involvement in both Aromhum scrotal hernias and human inguinal hernias. Conclusion: Our research underscores the central role of E2 in skeletal muscle fibrosis. Notably, we demonstrated that fibrosis can be entirely reversed by modulation of ESR1 signaling. Our study offers valuable insights into downstream genes and pathways that may serve as therapeutic targets for inguinal hernias and other fibrotic disorders. Funding: NIH: R01DK121529, Department of Veterans Affairs Research Career Scientist Award: IK6 RX003351. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.